Answers to today's fundamental biomedical questions generally require the application of techniques and methodologies from multiple scientific disciplines. In this RIMI collaborative project, Drs. Macher and Kaplan will utilize a range of research tools and approaches, incmuding genetics, molecular biology, cell biology, and MS to identify and characterize proteins that have critical functions in the kinetochore complexes and the ceil cycle. Their preliminary results have demonstrated that Sgt1p forms transient interactions with the kinetochore complexes and that it is post transtationally modified by phosphorylation, in the proposed research, further studies will be executed to determine the importance of the phosphoryiation sites already identified, and using the new mass spectrometer (i.e. MALDI-TOF) that will be available through the proposed Protein Chemistry facility, determine if there are additional sites of phosphorylation, and their location in the Sgt1p sequence. Genetic experiments provide evidence that SGT1 regulates the transition of ceils through the yeast "Start" point and the assembly of kinetochore complex. To determine whether SGT1 regulates these processes via a common pathway, or through multiple distinct pathways, we propose to analyze proteins associated with Sgt1p during the course of the cell cycle and under specific growth conditions: This will be accomplished by a combination of immunoprecipitation and proteomics (i.e. MS and database searching). The results of these studies will not only provide new insights into the regulation of the cell cycle and its relationships with kinetochore complex assembly and disassembly, but also identify potential targets for cancer drug design. As importantly, students from SFSU will gain valuable experience in a range of research topics and techniques that otherwise would not be available without this collaboration.